The major thrust of the work to be performed will be to develop new toxin effector moieties to be conjugated to monoclonal antibodies, with the goal of creating more efficacious immunotoxins than those formed with isolated A chains. Cloning and mutagenesis of diphtheria toxin will be primary tools. We shall seek expression of cloned fragments of the toxin at sufficiently high levels so as to be able to isolate milligram quantities of the expressed polypeptides. To this end, we will be using shuttle vectors to clone into B subtilis and will be modifying control regions of the tox gene to enhance efficiency of expression in E. coli. Another important step will be to devise ways to obtain forms of whole toxin modified at specific sites in the B moiety. Procedures to accomplish this without the necessity of cloning the whole toxin gene (permissible only under P4 conditions) are outlined. In addition to the work on the cloned diphtheria toxin gene, we will be attempting to clone the gene for Pseudmonas aeruginosa exotonin A, a toxin related to diphtheria toxin, and will work towards mutagenesis of the receptor binding site of this toxin. Finally, we shall be continuing the work on conjugates of the dimer of diphtheria toxin and chemically modified forms of diphtheria toxin.